1. Field of the Invention
The present invention relates to a method for determining a white blood cell count of a whole blood sample. More particularly, the present invention is concerned with a method for determining a white blood cell count of a whole blood sample, which comprises: mixing a whole blood sample with a surfactant to thereby obtain a mixture; allowing the mixture to stand for a time sufficient to lyze the white blood cells contained in the whole blood sample and release intrinsic myeloperoxidase from the white blood cells; measuring the concentration of the released myeloperoxidase in the mixture; and determining the white blood cell count in the whole blood sample, based on the, concentration of the released myeloperoxidase. By the method of the present invention for determining a white blood cell count, the determination of a white blood cell count and/or a neutrophil count of a whole blood sample can be performed easily and rapidly without the need to separate blood cells from the whole blood sample. Further, by the determination method of the present invention, in addition to a white blood cell count of a whole blood sample, the concentration of C-reactive protein contained in the same whole blood sample can be measured, so that the presence or absence of an infectious disease and the graveness of an inflammation can be diagnosed rapidly and easily and at low cost.
2. Prior Art
In recent years, in the initial steps of diagnosis of patients, as examination items for judging whether or not patients have a bacterial infectious disease, the white blood cell count of a whole blood sample and the concentration of C-reactive protein (hereinafter, frequently referred to simply as xe2x80x9cCRPxe2x80x9d) contained in a whole blood sample are determined. In this connection, for enabling doctors to take adequate measures, such as the administration of an antibiotic, it has been desired to develop determination techniques which can produce results rapidly and easily and at low cost.
Usually, the determination of the white blood cell count is performed using a commercially available automated blood cell counting apparatus which is based on the aperture-impedance method, wherein the apparatus is represented by COULTER COUNTER(trademark) (manufactured and sold by COULTER ELECTRONICS, INC., the U.S.A.). However, the concentration of CRP, which is a plasma protein, cannot be determined by such an apparatus.
Recently, there has been put into the market an apparatus for determining both the white blood cell count and the CRP concentration, which apparatus is described in Yasuo YAMAO, Hiroshi OKUNARI and Henri CHAMPEIX: Readout, 16, 11-15 (1998), wherein, in the apparatus, a blood cell counting apparatus based on the aperture-impedance method is given a CRP determination function based on a latex turbidimetric immunoassay. In the case of the use of such apparatus, the procedure is as follows: the red blood cells in the whole blood sample are first lyzed, and, then, the white blood cell count is determined by the aperture-impedance method, and, subsequently, the CRP determination is performed by a method in which the blood sample containing the lyzed blood cells is reacted with latex beads having bound thereto an anti-CRP antibody for a predetermined time, and then the CRP concentration is determined by a latex turbidimetric immunoassay. That is, in the case of the use of the above-mentioned apparatus, the determination of the white blood cell count and the determination of the CRP concentration are performed based on different principles, so that completely different operations are necessary for the determination. Therefore, the conventional method using the above-mentioned apparatus has drawbacks in that very cumbersome operations are necessary and that a number of reagents are used for the determination, thus leading to an increase in cost. In addition, the maintenance of the apparatus needs much time and large cost. Due to these drawbacks, an automated blood cell counting apparatus has not yet been introduced in almost all of medical institutions which have the responsibility of providing primary care, such as practicing physicians, and general hospitals, and therefore, the needs for medical care cannot be fully satisfied.
As a means to solve the above-mentioned problems, conceivable is a method in which both the white blood cell count and the CRP concentration are determined, based on the same principle. Specifically, conceivable is a method in which, with respect to a single sample of whole blood, both the white blood cell count and the CRP concentration are determined by an immunological method. An immunological determination of the white blood cell count can be made by measuring a protein which is specifically present in white blood cells.
It is generally know that, when a bacterial infection or an inflammation occurs, the number of neutrophis in the blood is increased, wherein the neutrophis have a largest proportion among all types of white blood cells. Therefore, by determining the neutrophil count, the increase and decrease in the number of white blood cells can be detected, so that the presence or absence of a bacterial infectious disease and the graveness of an inflammation can be judged.
As an example of a method for measuring the neutrophil count of an inflamed site of tissue or measuring the white blood cell count or the granulocyte count of a whole blood sample, there can be mentioned the method described in Peter P. Bradley, Dennis A. Priebat, Robert D. Christensen and Gerald Rothstein: J. Invest. Dermatol., 78, 206-209 (1982). This method is as follows. First, neutrophils are separated from a whole blood sample by using the Ficoll reagent, or an inflamed skin tissue is obtained. The obtained neutrophils or the obtained inflamed skin tissue is homogenized in a 50 mM potassium phosphate buffer solution (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide (HTAB) and then sonicated to extract myeloperoxidase, and subsequently the enzyme activity of the extracted myeloperoxidase is determined.
Another method is disclosed in Rita Cramer, Maria Rosa Soranzo, Pietro Dri, Renzo Menegazzi, Anna Pitotti, Giuliano Zabucchi and Pierluigi Patriarca: Journal of immunological Methods, 70, 119-125 (1984). This method is as follows. Dextran (MW 200,000) is added to blood containing a solution of ACD (acid citrate dextrose) as an anticoagulant, to obtain a mixture. From the obtained mixture, red blood cells are removed. Subsequently, the Ficoll reagent is added to the resultant, and granulocytes are separated from the resultant mixture by centrifugation. Then, 2xc3x97104 cells of the granulocytes are mixed with a 0.1 M phosphate buffer solution (pH 7.0) containing 13 mM guaiacol and 0.02% cetyltrimethylammonium bromide. 1 xcexcmol of H2O2 is added thereto, and the peroxidase activity is measured.
W. M. Kuebler, C. Atels, L. Schuerer and A. E. Goetz: Int. J. Microcirc., 16, 89-97(1996) disclose the following method. Polymorphonuclear white blood cells are separated from whole blood by using the Ficoll reagent. The obtained white blood cells are lyzed with a 50 mM potassium phosphate buffer solution (pH 6.0) containing 0.5% HTAB, and then the enzyme activity of the myeloperoxidase in the resultant cell lysis mixture is determined. Further, the above-mentioned prior art document also discloses a method in which rat brain tissue or rabbit lung tissue is homogenized in 1 ml of a 0.02 M potassium phosphate buffer solution (pH 7.4) which is cooled with ice, and then 1 ml of 0.02 M potassium phosphate buffer solution (pH 7.4) is added thereto, and the resultant is centrifuged at 2,000 rpm for 15 minutes at 4xc2x0 C. to obtain a centrifugation product containing a supernatant. Subsequently, the enzyme activity of the myeloperoxidase in the supernatant is determined. Further, the residue of the centrifugation product is incubated at 60xc2x0 C. for 2 hours, and then homogenized with a 50 mM potassium phosphate buffer solution (pH 6.0) containing 0.5% HTAB, and then sonicated, and the resultant is centrifuged at 20,000 rpm for 15 minutes at 4xc2x0 C. to obtain a supernatant. Subsequently, the enzyme activity of the myeloperoxidase in the supernatant is determined.
International application publication No. WO93/01306 discloses the following method. A predetermined amount of blood is put on a glass microfiber filter set in a syringe. Water is added to the blood, and the red blood cells are lyzed without breaking the granulocytes, and the cell lysis components in the blood are removed by pressure washing, to thereby remove components hindering the determination of the granulocyte count and obtain non-lyzed granulocytes carried on the glass microfiber filter. The glass microfiber filter carrying the non-lyzed granulocytes is dried, and a 50 mM phosphate buffer solution containing 0.005% Triton(trademark) X-100, 30 mM 3-amino-1,2,4 triazole, 0.0005% H2O2 and 0.5 mg/ml o-tolidine is dropped on the glass microfiber filter, so that myeloperoxidase is released from the granulocytes and forms blue spots by staining. The blue spots are counted as representing the granulocytes.
Japanese Patent Application Laid-Open Specification No. 8-308593 (corresponding to EP 743519 and U.S. Pat. No. 5,639,630) discloses the following method. A solution is prepared which comprises: a nonionic polyethoxylate surfactant, an anionic surfactant or an amphoteric surfactant in a concentration which lyzes the red blood cells in whole blood thereby releasing hemoglobin from the red blood cells but which does not break the white blood cells; formaldehyde or paraformaldehyde in a concentration which chemically crosslinks the white blood cells but which does not crosslink the red blood cells; a sugar or a sugar alcohol which enhances the detectablility of the lymphocytes; and a buffer solution which causes the reaction mixture to have a pH value which is at or around the neutral point, specifically a pH range of from about 6.8 to about 8.0. The solution is mixed with whole blood, and the resultant mixture is heated to about 60 to 75xc2x0 C. to lyze the red blood cells and cross-link the white blood cells. A part of the white blood cells are stained using the intrinsic peroxidase activity, and cell counts of the different types of white blood cells are determined by electro-optical analysis using absorbance or light scattering.
However, these methods have problems as follows. Since myeloperoxidase is substantially released from neutrophils present in the tissues of the skin, the brain and the lung, and subjected to the determination of the enzyme activity, it is necessary to conduct extremely cumbersome operations, such as homogenization treatment in the presence of HTAB and centrifugation treatment, and much time is also needed. Further, in determining the white blood cell count of whole blood, in order to remove substances which hinder the measurement of myeloperoxidase, it is necessary to conduct an operation in which the white blood cells are separated from whole blood by using the Ficoll reagent or the like. Therefore, it is necessary to conduct the operation for removing substances which hinder the measurement and to perform a reaction for fixing the white blood cells, so that the procedure becomes cumbersome and it is necessary to use a number of reagents and special apparatuses.
Akiko GOTO, Ichio UCHIDA, Hitoshi TOMITA: xe2x80x9cRinsho Kensa (Clinical Examinations)xe2x80x9d, 40, 859-863 (1996) and Japanese Patent Application Laid-Open Specification Nos. 8-145993, 9-72906, 9-196919 and 11-32793 disclose a method for the counting of the white blood cells contained in the urine of a patient of a urinary tract infection. Specifically, in this method, a urine sample is treated with 0.05% Triton(trademark) X-100, and the concentration of myeloperoxidase released from the white blood cells (95% of them are neutrophils) contained in the urine sample is measured by enzyme immunoassay.
The amount of cellular components in urine is extremely small, as compared to the amount of cellular components in blood. Therefore, in urine, the amount of substances which hinder the measurement of the concentration of myeloperoxidase is small. For this reason, in the above-mentioned method, a urine sample is treated only with Triton(trademark) X-100 before the measurement of the concentration of myeloperoxidase released from the neutrophils contained in the urine sample. However, whole blood contains a mixture of white blood cells, red blood cells, platelets and plasma proteins and, hence, whole blood is a system containing various types of biological components in high concentrations. Therefore, it has been inconceivable that such method suitably applicable to a urine sample can be applied to whole blood.
There has been a report of the study in which whole blood is subjected to lysis treatment to thereby release peptides called xe2x80x9cdefensinsxe2x80x9d from the neutrophils in the whole blood, and the concentration of the released defensins is measured, and correlations between defensins and the white blood cell count and the neutrophil count are investigated (see Toshihiko Ihi, Masamitsu Nakazato, Hiroshi Mukae and Shigeru Matsukura: Clinical Infection Diseases, 25, 1134-1140 (1997)). However, in the method used in the abovementioned study, it is necessary to perform a cumbersome procedure that 1 M acetic acid is added to whole blood and the resultant mixture is homogenized by means of a polytron homogenizer, and the resultant is centrifuged under 2,000xc3x97g for 30 minutes at 4xc2x0 C. thereby extracting the peptide. Therefore, this method cannot be practically used at all at sites of medical service where it is necessary to rapidly and easily determine the white blood cell count of a whole blood sample.
As described hereinabove, in the conventional methods for immunologically determining a substance derived from white blood cells, it is necessary to perform cumbersome and time-consuming operations, such as an operation in which the white blood cells are separated from whole blood, and an operation in which the white blood cells are subjected to lysis treatment under stringent conditions for a long time in order to substantially release a desired substance from the white blood cells. Further, the conventional methods have a problem in that the determined value is not necessarily well in agreement with a white blood cell count as directly measured by means of a blood cell counting apparatus.
International Patent Application Publication No. WO99/46599 (corresponding to Japanese Patent Application Laid-Open Specification No. 11-258231) (which was published after the filing date of the basic Japanese patent application of the present application) discloses a method in which a surfactant is added to a whole blood sample thereby releasing intrinsic elastase from the granulocytes, and the released elastase is measured by an immunological method using xcex11-antitrypsin inhibitor, which is an inhibitor against elastase, and the white blood cell count is determined, based on the concentration of the released elastase. However, in this method, for measuring the granulocyte elastase, it is necessary to perform an operation of adding xcex11-antitrypsin inhibitor. Therefore, this method is disadvantageous in that the procedure is cumbersome and the cost becomes high. Further, although a good correlation is present between the granulocyte elastase and the white blood cell count, the above-mentioned document has no description that this method is applied to a whole blood sample having a white blood cell count of 10,000 cells/xcexcl or more, i.e., a whole blood sample obtained from a non-healthy human.
Thus, by any of the conventional methods, it is impossible that an intrinsic substance of the white blood cells is measured rapidly and easily by an immunological method and a white blood cell count of a whole blood sample is accurately determined without the need to separate the white blood cells from the whole blood sample. Also, by any of the conventional methods, it is impossible that, in addition to a white blood cell count of a whole blood sample, the concentration of CRP contained in the same whole blood sample is measured rapidly and easily.
The present inventors have made extensive and intensive studies with a view toward solving the above mentioned problems. As a result, it has surprisingly been found that the above object can be attained by a determination method in which a whole blood sample is mixed with a surfactant to thereby efficiently lyze the white blood cells and release intrinsic myeloperoxidase from the white blood cells, and the concentration of the released myeloperoxidase is measured. Specifically, it has unexpectedly been found that the myeloperoxidase concentration as measured by the above method exhibits a very good correlation with a white blood cell count as measured by the conventional aperture-impedance method, so that a white blood cell count can be determined, based on the myeloperoxidase concentration. Further, it has also been found that, in the above method, the concentration of C-reactive protein contained in the whole blood sample can also be measured in addition to the myeloperoxidase concentration. The present invention has been completed, based on these novel findings.
Therefore, it is a primary object of the present invention to provide a method for determining a white blood cell count of a whole blood sample rapidly and easily and at low cost.